The human immune system comprises numerous different types of cells having overlapping functions which together act to protect the human body against sickness and disease. The cells of the immune system have complex multiple functions and interconnecting relationships.
GM-CSF, IL-3, and IFN-.gamma. are all cytokines. "Cytokines" are a class of compounds which regulate responses of cells of the immune system, such as B and T lymphocyte cells ("B cells" and "T cells") and natural killer ("NK") cells. A "cytokine" is a generic term for a nonantibody protein released by certain cell populations on contact with an inducer and which acts as an intercellular mediator. A "lymphokine" is a soluble substance released by sensitized lymphocytes on contact with specific antigen or other stimuli which helps effect cellular or humoral. immunity.
The terms "cytokine" and "lymphokine" have become interchangeable. In an attempt to simplify the nomenclature of these compounds, a group of participants at the Second International Lymphokine Workshop held in 1979 proposed the term "interleukin," abbreviated "IL," to develop a uniform system of nomenclature based on the ability of the proteins to act as communication signals between different populations of leukocytes.
To date, 21 different cytokines, most but not all of which are produced by T cells, have been identified. Each has a distinct molecular configuration and performs a different task. A number of the known cytokines have been shown to have a demonstrable activity on B cells. In vitro, the lymphokines IL-1, IL-2, IL-4, IL-5, IL-6, IL-10, IFN-.gamma., and TGF-.beta. (transforming growth factor .beta.) have been shown to enhance B cell proliferation, immunoglobulin secretion, or to otherwise play a role in influencing the subclass of secreted Ig. Depending on the system being studied, addition of either one or a number of the above lymphokines has been shown to increase in vivo antibody production or to alter the isotype (i.e., IgG, IgM, IgE, IgA, etc.) of secreted antibody. Among the lymphokines reported to influence B cell proliferation include IL-1, IL-2, IL-4, and IL-10, and those reported to influence B cell differentiation and Ig secretion include IL-2, IL-5, IL-6, TGF-.beta., and IFN-.gamma..
None of the reported cytokines which enhance Ig secretion in vitro have been shown to play a prominent role in vivo. Thus, infusion of monoclonal antibodies specific for IL-2, IL-5, IL-6, or IFN-.gamma. does not significantly suppress antigen-stimulated antibody production. This suggests that, under physiologic conditions, B cell differentiation depends solely on direct T cell interaction, or that other as yet unknown cytokines mediate this step.
While immune responses to antigens that stimulate T cell activation (the so-called T dependent antigens or "TD antigens") could rely on direct T cell interactions with B cells to effect Ig secretion, this is not the case with antigens that are unable to induce T cell activation. Antigens which are T cell independent ("TI antigens") induce high levels of antibody production in the absence of direct or even indirect T cell help. Thus, the events that regulate B cell differentiation and immunoglobulin secretion to TI antigens must rely on other as yet undefined pathways. Since B cell differentiation leading to immunoglobulin secretion is the final event which underlies a competent humoral antibody system, defining the events or cytokines which regulate this step is invaluable in designing methods for amplifying or suppressing an immune response.
To facilitate a quick appreciation of the invention, the following provides a brief description of the primary known functions of immunoglobulin, antibodies, lymphocytes, B cells, T cells, and NK cells as background. Also provided is a brief summary of the known activities of the cytokines reported to influence B cell proliferation or antibody secretion, namely IL-1, IL-2, IL-4, IL-5, IL-6, IL-10, TGF-.beta., and IFN-.gamma.. A summary of the known activities of GM-CSF and IL-3 is also provided. Reference materials include Fundamental Immunology, Second Edition, William E. Paul, M. D., ed. (Raven Press, New York 1989); Fundamental Immunology, Third Edition, William E. Paul, M.D., ed. (Raven Press, New York 1993); Interferon: Principles and Medical Applications, S. Baron et al., eds. (The University of Texas Medical Branch at Galveston, Galveston, Texas 1992); The Cytokine Handbook, Angus Thomson, ed. (Academic Press Inc., San Diego, Calif. 1992); and The Cytokine Handbook, Second Edition, Angus Thomson, ed. (Academic Press Inc., San Diego, Calif. 1994), all of which are specifically incorporated by reference.
Mammals, including man, are confronted on a daily basis with a myriad of organisms. A major component of the immune system that plays an essential role in protecting the host against infection by these organisms is the humoral antibody. Antibodies are protein molecules, also known as immunoglobulins, which have exquisite specificity for the foreign particle which stimulates their production. For example, systemic infection with "bacteria A" will induce antibodies that bind with a high avidity to "bacteria A" but not to "bacteria B." Similarly, "bacteria B" will induce anti-"bacteria B" antibodies which do not cross-react with "bacteria A."
Immunoglobulin (Ig) is a class of structurally related proteins consisting of two pairs of polypeptide chains, one pair of light (L) low molecular weight! chains (.kappa. or .lambda.), and one pair of heavy (H) chains (.gamma., .alpha., .mu., .delta., and .epsilon.), all four linked together by disulfide bonds. Both H and L chains have regions that contribute to the binding of antigen and that are highly variable from one Ig molecule to another. In addition, H and L chains contain regions that are nonvariable or constant. On the basis of the structural and antigenic properties of the H chains, Ig's are classified as IgG, IgA, IgM, IgD, and IgE isotypes. Subclasses of IgG's, based on differences in the H chains, are referred to as IgG1, etc.
Lymphocytes are white blood cells formed in lymphatic tissues throughout the body, such as lymph nodes, spleen, thymus, tonsils, Peyer's patches (small intestine tissue), and sometimes in bone marrow. Individual lymphocytes are specialized in that they are committed to respond to a limited group of structurally related antigens. This commitment, which exists prior to the first contact of the immune system with a given antigen, is expressed by the presence of antigen-specific receptors (i.e., immunoglobulin) on the lymphocyte membrane. The ability of an organism to respond to virtually any antigen is achieved by the existence of a very large number of different clones of lymphocytes, each bearing receptors specific for distinct antigens. In consequence, lymphocytes are an enormously heterogeneous collection of cells.
Lymphocytes differ from one another not only in the specificity of their receptors but also in their functional properties. Two broad classes of lymphocytes are recognized: the B lymphocytes and the T lymphocytes. In addition to these. two classes, lymphoid cells that mediate certain "nonspecific" cytotoxic responses are known. These include natural killer (NK) cells.
B lymphocytes, also known as "B cells," are a type of lymphocyte that derive from hematopoietic stem cells by a complex set of differentiation events that are only partially understood. B cells are precursors of antibody-secreting cells and thus are responsible for the production of immunoglobulins. The cell-surface receptor of B cells is an antibody or immunoglobulin (Ig) molecule specialized for expression on the cell surface. Newly differentiated B cells initially express surface Ig solely of the IgM class. Associated with maturation of a B cell is the appearance of other immunoglobulin isotypes on the surface of the B cell.
To release antibody in response to cytokines, the B cells must first be activated. There are many ways to activate B cells, including cross-linkage of membrane Ig molecules by the antigen (cross-linkage-dependent B cell activation), direct encounter with T cells (helper T cells or helper T cell-associated molecules, such as, for example, CD40 ligand), or encounter with mitogens. In such encounters, the antigen presents epitopes recognized by the B cell's cell-surface Ig.
Because each B cell bears multiple membrane Ig molecules with identical variable regions, optimal membrane-Ig mediated cross-linkage activation is achieved by a high level of cross-linkage of the cell-surface receptors, which requires that the antigen present more than one copy of the epitope that the cell-surface Ig recognizes. Although many simple protein antigens do not have this potential, such a requirement is fulfilled by polysaccharides and other antigens with repeating epitopes, such as surfaces of microbes and DNA. Among these antigens are the capsular polysaccharides of many medically important microorganisms, such as pneumococci, streptococci, and meningococci.
There are much data to show that cross-linkage of membrane Ig can also lead to elimination or inactivation of B cells. In general, it is believed that certain types of receptor cross-linkage events, if they occur in the absence of specific stimulatory signals, lead to inactivation rather than activation. The highly repetitive epitopes expressed on polysaccharides may lead to activation in the absence of costimulation, possibly because of the magnitude of the receptor-mediated stimulation.
T lymphocytes, or "T cells," are thymocyte derived, of immunological importance that is long-lived (months to years), and are responsible for cell-mediated immunity. T cells consist of functionally different populations, known as "helper T cells," "suppressor T cells," and "killer T cells." T cells involved in delayed hypersensitivity and related immune phenomena are also known.
Natural killer cells, or "NK cells," are lymphoid cells that mediate certain "nonspecific" cytotoxic responses. Such nonspecific cytotoxic responses kill certain forms of tumor cells using recognition systems that are different from those used by T or B cells. Killing of one cell type by another through contact interaction constitutes a major effector arm of self-defense of the immune system.
In addition to these cells, other compounds, the cytokines, play a significant role in protecting a host. One group of cytokines is the interleukins.
IL-1 is primarily an inflammatory cytokine, whereas IL-2 and other cytokines are primarily growth factors for lymphocytes. IL-1 is a polypeptide hormone synthesized by monocytes. During inflammation, injury, immunological challenge, or infection, IL-1 is produced and, because of its multiple biological properties, this cytokine appears to affect the pathogenesis of the disease. In animals, IL-1 is a potent. inducer of hypotension and shock. IL-1 acts on the hypothalamus to induce fever and directly on skeletal muscle to promote protein catabolism.
IL-2, also known as T cell growth factor, is a lymphokine and polypeptide hormone produced by both T helper and suppressor lymphocytes. This cytokine has direct effects on the growth and differentiation of T cells, B cells, NK cells, lymphokine-activated killer (LAK) cells, monocytes, macrophages, and oligodendrocytes.
IL-3, also known as multicolony stimulating factor, acts on numerous target cells within the hemopoietic system. This cytokine has the broadest target specificity of any of the haematopoietic growth factors (HPGFs), and can stimulate the generation and differentiation of hemopoietic stem cells (i.e., precursors of blood cells), which give rise to macrophages, neutrophils, eosinophils, basophils, mast cells, megakaryocytes, and erythroid cells.
The relationship between IL-3 and B cells was unclear prior to the invention. In fact, as of 1994, it was believed that the range of target cells of IL-3 did not include cells committed to the T- and B-lymphoid lineages, and that there was no compelling evidence that IL-3 had a significant, direct effect on B-cell development. J. W. Schrader, "Chapter 5: Interleukin-3," The Cytokine Handbook, 2nd Ed., Angus Thomson, ed., page 84 (Academic Press, New York, 1994).
Secretion of IL-3 by B cells has not been reported, although IL-3 is synthesized by T cells and mast cells. Several reports demonstrated that IL-3 could induce a modest enhancement of Ig secretion by human B cells activated with SAC (a polyclonal activator) and IL-2. For example, Xia et al., "Human Recombinant IL-3 is a Growth Factor for Normal B Cells," J. of Immunology, 148, 491-497 (1992), reported that IL-3 enhanced the proliferation of a population of cells enriched in B cells. Similarly, Tadmori et al., "Human Recombinant IL-3 Stimulates B Cell Differentiation," J. of Immunol., 142, 1950-1955 (1989), reported that IL-3 stimulated IgG secretion from tonsillar cells containing B cells or in a population of peripheral blood-derived enriched B cells activated by bacterial antigen. In addition, Matsumoto et al., "Induction of IgE Synthesis in Anti-IgM-Activated Nonatopic Human B Cells by Recombinant Interleukin-3," Int. Arch. Allergy Appln. Immunol., 89, 24-30 (1989), reported that human recombinant IL-3 augmented IgE synthesis by normal B cells or mixtures of T and B lymphocytes, and that IL-1, IL-2, IL-5, IL-6, GM-CSF, G-CSF, M-CSF, and IFN-.gamma. failed to induce IgE synthesis. Matsumoto et al. also note that they could not conclusively identify IL-3 as the factor in the T cell supernatant responsible for inducing IgE synthesis because the activity could not be reversed by the addition of anti-IL-3 antibody.
These results were attributed to an IL-3-mediated enhancement in cell growth. In these studies, B cells were not electronically sorted and thus were not highly purified. Thus, the Ig enhancing effect may reflect the action of IL-3 on many contaminating non-B, non-T cells in the population, and it is therefore not possible to determine from these experiments whether IL-3 was acting directly on the B cell. Further, in prior experiments, B cells were not fractionated according to size. Thus, a possible role for the prior activational state of the B cell was not addressed.
In further contrast to the present invention, Kimoto et al., "Recombinant Murine IL-3 Fails to Stimulate T or B Lymphopoiesis In Vivo, But Enhances Immune Responses To T Cell-Dependent Antigens," J. of Immunology, 140, 1889-1894 (1988), reported that mice bearing osmotic minipumps loaded with murine recombinant IL-3 showed no increase in the lymphoid organs of the total number of B and T cells. Furthermore, Kimoto et al. suggested that IL-3 does not act directly on lymphocytes or their precursors, but may potentiate the humoral immune response to T cell-dependent antigens, presumably by acting on accessory cells.
IL-4 is a glycoprotein also known as B cell stimulating factor 1 (BSF-1) and B cell differentiation factor. It functions to costimulate B cell growth, Ig class switching, T cell growth and differentiation, macrophage activation, regulate mast cell growth, and to costimulate hematopoietic precursor cells.
IL-5, also known as B cell growth factor II (BGF-II), T cell replacing factor, IgA-enhancing factor, and eosinophil colony stimulating factor, is a glycoprotein produced by T lymphocytes and mast cells. This cytokine has the dual functions of a colony stimulating factor, as well as promoting the differentiation of eosinophilic colonies in bone marrow. IL-5 induces specific in vitro antibody production by B cells primed with antigen in vivo. While IL-5 serves as a differentiation factor in vitro, it does not appear to act as a differentiation factor in vivo.
IL-6, also known BSF-II, interferon .beta.2, hybridoma/plasmacytoma growth factor, and hepatocyte stimulatory factor, is a glycoprotein produced by both lymphoid and nonlymphoid cells. This cytokine regulates immune responses, acute-phase reactions, and hemopoiesis. IL-6 acts on B cell lines at the mRNA level and induces biosynthesis of secretory-type Ig. In addition to IL-5, IL-6 has also been shown under very restricted conditions to function as a differentiation factor. All other known T cell or macrophage derived factors that have been tested cannot induce activated B cells to secrete Ig in the absence of added growth factors.
IL-10, also known as cytokine synthesis inhibitory factor, is produced by T cells, macrophages, and other cell types. This cytokine inhibits several macrophage functions, including cytokine synthesis and some microbial activities, in addition to enhancing or stimulating mast cells and B cells. IL-10 causes strong proliferation of human B cells activated by anti-CD40 antibodies or cross-linking of the antigen receptor.
In addition to the interleukins, other cytokines have been characterized. Colony stimulating factors (CSFs) are a group of factors primarily concerned with hematopoiesis. They are defined as proteins which stimulate the clonal growth of bone-marrow cells in vitro.
Granulocyte-macrophage colony stimulating factor (GM-CSF) is a glycoprotein growth factor that modulates the growth or differentiation of hemopoietic cells. This growth factor can be produced by a number of different cells under different circumstances, including T cells, macrophages, endothelial cells, stromal cells, fibroblasts, mast cells, and others. The major actions of GM-CSF involve the regulation of survival, differentiation, and proliferative and functional activities in granulocyte-macrophage populations. There are no reports prior to the invention indicating that GM-CSF can stimulate the release of antibody by B cells.
Finally, another class of cytokines that function in the body's immune system is interferons (IFNs). IFNs are major contributors to the first line of antiviral defense by inhibiting virus replication, in addition to exerting many other important effects on cells. IFNs do not act directly to protect cells from infection. Rather, they stimulate production of a protein in neighboring cells that stops the growth of the virus, thus protecting the cells from infection.
IFNs are classified into three groups, alpha, beta, and gamma, based on the cells of origin and method of induction. The production of IFN-.alpha. and IFN-.beta. is not a specialized cell function, and probably all cells of the organism are capable of producing these IFNS.
In contrast to IFN-.alpha. and IFN-.beta. synthesis, which can occur in any cell, production of IFN-.gamma. is a function of T cells and NK cells. All IFN-.gamma. inducers activate T cells either in a polyclonal (mitogens or antibodies) or in a clonally restricted, antigen-specific manner. Human IFN-.gamma. promotes proliferation of activated human B cells and, in cultures of human B cells, can act synergistically with IL-2 to enhance immunoglobulin light-chain synthesis.
The brief discussion describing functions of various human immune cells, and the known activities of IL-1, IL-2, IL-3, IL-4, IL-5, IL-6, IL-10, GM-CSF, and IFN-.gamma. exemplifies the extreme diversity of the human immune system. Despite this level of knowledge, however, there is no complete understanding of the intricacies of the immune system. Tremendous gaps remain. For example, it is not possible to make general statements about the properties of cytokines, except that they act as intercellular mediators by regulating responses of cells of the immune system. Thus, there remains a need in the art for a greater understanding of the immune system and for the provision of additional and superior methods of treating immune disorders.